Non-adenoviral gene product-based complementing cells for adenoviral vectors

ABSTRACT

The invention provides cells and methods of using the cells for the propagation of replication-deficient adenoviral vectors. The cells comprise at least one heterologous nucleic acid sequence which upon expression produces at least one non-adenoviral gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome so as to propagate a replication-deficient adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the one or more regions when present in the cell.

FIELD OF THE INVENTION

This invention pertains to cells for the propagation of adenoviralvectors.

BACKGROUND OF THE INVENTION

Recombinant eukaryotic viral vectors have become a preferred means ofgene transfer for many researchers and clinicians. The human adenovirusis one of the most widely used recombinant viral vectors in current genetherapy protocols. As the use of adenoviral vectors becomes moreprevalent, the need for systems that efficiently produce adenoviralvectors suitable for administration is increasingly important.

A concern associated with recombinant adenoviral vectors is uncontrolledpropagation of the vector upon administration. To address this concern,replication-deficient adenoviral vectors, typically lacking theessential E1 region of the adenoviral genome, have been developed.

The production of replication-deficient adenoviral vectors is commonlyaccomplished by use of a complementing cell line, such as the 293 cellline developed by Graham et al. (J. Gen. Virol., 36, 59-72 (1977)),which provides in trans the gene functions lacking in thereplication-deficient adenoviral vector. A problem associated with manycomplementing cell lines, including the 293 cell line, is thepossibility of homologous recombination between thereplication-deficient adenoviral genome and the regions of theadenoviral genome inserted into the complementation cell, resulting inproduction of replication-competent adenovirus (RCA). To reduce thefrequency of RCA formation, several researchers have attempted toconstruct complementing cell lines comprising viral gene sequences thatlack any homology to the adenoviral vector of interest (see, forexample, International Patent Applications WO 94/28152 and WO 98/39411,and U.S. Pat. No. 5,994,128 and 6,033,908).

The construction of stable human cell lines that effectively andefficiently complement replication-deficient viral vectors can bedifficult. For example, such cell lines often produce significantquantities of E1 and/or E4 gene products, resulting in undesiredcytotoxic and/or cytostatic effects. High levels of E1A gene productexpression, for example, induce apoptosis in host cells (Rao et al.,PNAS, 89, 7742-7746 (1992)), while expression of E4 gene products inducep53-independent apoptosis in human cells (Marcellus et al., J. Virol.,72, 7144-53 (1998)). Thus, complementation cells, such as those known inthe art, that constitutively express such factors may be associated withpoor survival rates prior to and/or during adenoviral vector production.

Animal cells and other viruses encode gene products that arefunctionally homologous to adenoviral early region genes. Some of thesegene products, when transiently expressed in human primary cells ornon-human transformed cells, have been shown to complement fordeficiencies in E1A gene functions. In particular, Tevethia et al.,Virology, 161, 276-285 (1987), describes complementation in primaryembryonic lung cells of an E1A-deleted adenoviral vector with plasmidsencoding immediate early region genes of the human cytomegalovirus(CMV). In addition, the E7 protein of human papilloma virus 16 (HPV16)complements for deficiencies in the E1A region by immortalizing primaryrat cells, as shown by co-infection experiments, while tamarin cellstransformed by the Epstein-Barr virus (EBV) complement for deficienciesin the E1A and/or E2 regions (see, e.g., Kimura et al., Tumor Research,32, 1-21 (1997), and Horvath et al., Virology, 184, 141-148 (1991)).Moreover, some cell lines, including the human hepatoblastoma line HepG2and certain embryonic stem cell lines, encode factors that provide forthe transcriptional transactivation function of the E1A region, as shownby activation of both E2A and/or E1B promoters in the absence of the E1Aregion (see, e.g., Spergel et al., Proc. Natl. Acad. Sci. USA, 88,6472-6476 (1991), Spergel et al., J. Virol., 66, 1021-1030 (1992),Imperiale et al., Mol. Cell Biol., 4, 867-874 (1984), La Thangue andRigby, Cell, 49, 507-513(1987), and La Thangue et al., Nuc. Acids Res.,18, 2929-2938 (1990)).

Accordingly, there remains a need for alternative cells for propagatingreplication-deficient adenoviral vectors. The invention provides suchcells. These and other advantages of the invention, as well asadditional inventive features, will be apparent from the description ofthe invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides a cell having a cellular genome comprising aheterologous nucleic acid sequence, which upon expression produces atleast one non-adenoviral gene product. The non-adenoviral gene productcomplements in trans for a deficiency in at least one essential genefunction of one or more regions of an adenoviral genome so as topropagate a replication-deficient adenoviral vector comprising anadenoviral genome deficient in the at least one essential gene functionof the one or more regions when present in the cell. The invention alsoprovides a transformed human cell comprising a heterologous nucleic acidsequence which upon expression produces at least one non-adenoviral geneproduct that complements in trans for a deficiency in at least oneessential gene function of one or more regions of an adenoviral genomeso as to propagate a replication-deficient adenoviral vector comprisingan adenoviral genome deficient in the at least one essential genefunction of the one or more regions when present in the cell.

The invention also provides a system comprising the inventive cell and areplication-defective adenoviral vector comprising an adenoviral genomedeficient in the at least one essential gene function of the one or moreregions. The invention further provides a method of propagating areplication-deficient adenoviral vector, wherein the method comprisesproviding the inventive cell, introducing a replication-deficientadenoviral vector into the inventive cell, and maintaining the cell topropagate the replication-deficient adenoviral vector.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a cell having a cellular genome comprising atleast one heterologous nucleic acid sequence, which upon expressionproduces at least one non-adenoviral gene product that complements intrans for a deficiency in at least one essential gene function of one ormore regions of an adenoviral genome of a replication-deficientadenoviral vector so as to propagate (i.e., replicate the entire lifecycle of, or replicate to any stage of the life cycle of) thereplication-deficient adenoviral vector when present in the cell.

The cell can be any suitable cell that comprises a genome that canincorporate and preferably retain the heterologous nucleic acid encodingat least one non-adenoviral gene product that complements in trans for adeficiency in at least one essential gene function of one or moreregions of an adenoviral genome. The cell desirably can propagateadenoviral vectors and/or adeno-associated viral (AAV) vectors wheninfected with such vectors or with nucleic acid sequences encoding theadenoviral or AAV genome. Most preferably, the cell can propagate asuitable replication-deficient adenoviral vector upon infection with anappropriate replication-deficient adenoviral vector or transfection withan appropriate replication-deficient viral genome.

Particularly desirable cell types are those that support high levels ofadenovirus propagation. The cell preferably produces at least about10,000 viral particles per cell and/or at least about 3,000 focusforming units (FFU) per cell. More preferably, the cell produces atleast about 100,000 viral particles per cell and/or at least about 5,000FFU per cell. Most preferably, the cell produces at least about 200,000viral particles per cell and/or at least about 7,000 FFU per cell.

Preferably, the cell is, or is derived from, an anchorage dependentcell, but which has the capacity to grow in suspension cultures. Morepreferably, the cell is a primary cell. By “primary cell” is meant thatthe cell does not replicate indefinitely in culture. Examples ofsuitable primary cells include, but are not limited to, human embryonickidney (HEK) cells, human retinal cells, and human embryonic retinal(HER) cells. Most preferably, the cells are human embryonic lung (HEL)cells or ARPE-19 cells. Alternatively, the cell can be a transformedcell. The cell is “transformed” in that the cell has the ability toreplicate indefinitely in culture. Examples of suitable transformedcells include renal carcinoma cells, CHO cells, KB cells, HEK-293 cells,SW-13 cells, MCF7 cells, and Vero cells. Preferably, the cell is a lungcarcinoma cell, such as, for example, a non-small cell lung carcinomacell. The non-small lung cell carcinoma cell can be asquamous/epidermoid carcinoma cell, an adenocarcinoma cell, or a largecell carcinoma cell. The adenocarcinoma cell can be an alveolar cellcarcinoma cell or bronchiolo-alveolar adenocarcinoma cell. Othersuitable non-small cell lung carcinoma cells include the cell linesNCI-H2126 (American Type Culture Collection (ATCC) No. CCL-256), NCI-H23(ATCC No. CRL-5800), NCI-H322 (ATCC No. CRL-5806), NCI-H358 (ATCC No.CRL-5807), NCI-H810 (ATCC No. CRL-5816), NCI-H1155 (ATCC No. CRL-5818),NCI-H647 (ATCC No. CRL-5834), NCI-H650 (ATCC No. CRL-5835), NCI-H1385(ATCC No. CRL-5867), NCI-H1770 (ATCC No. CRL-5893), NCI-H1915 (ATCC No.CRL-5904), NCI-H520 (HTB-182), and NCI-H596 (ATCC No. HTB-178). Alsosuitable are squamous/epidermoid carcinoma lines that include HLF-a(ATCC No. CCL-199), NCI-H292 (ATCC No. CRL-1848), NCI-H226 (ATCC No.CRL-5826), Hs 284.Pe (ATCC No. CRL-7228), SK-MES-1 (ATCC No. HTB-58),and SW-900 (ATCC No. HTB-59), large cell carcinoma lines (e.g., NCI-H661(ATCC No. HTB-183)), and alveolar cell carcinoma lines (e.g., SW-1573(ATCC No. CRL-2170)). The most preferred cell is selected from the groupconsisting of an A549 cell (ATCC CCL-185), an NCI-H1299 (ATCC CRL-5803)cell, a Calu-1 cell (ATCC HTB-54), and an NCI-H460 (ATCC HTB-177) cell.Alternatively, the transformed cell need not be a lung carcinoma cell.In this respect, the cell is preferably a HeLa cell (ATCC CCL-2) or anARPE-19/HPV-16 cell (ATCC CRL-2502). In addition, the transformed cellcan be any cell transformed by a viral gene isolated from anon-adenovirus family member, such as, for example, genes encoded byPapillomaviridae, Poxviridae, Polyomaviridae, Hepadnaviridae,Picorniviridae, Flaviviridae, or any other suitable virus family asdefined by van Regenmortel et al., eds., Virus Taxonomy, Seventh Reporton the International Committee on Taxonomy of Viruses, 2000.

The cell comprises at least one heterologous nucleic acid sequence asdescribed herein, i.e., the cell can comprise one heterologous nucleicacid sequence as described herein or more than one heterologous nucleicacid sequence as described herein (i.e., two or more of the heterologousnucleic acid sequences). Such cell lines can be generated in accordancewith standard molecular biological techniques as described inInternational Patent Application WO 95/34671 and U.S. Pat. No.5,994,106. The heterologous nucleic acid sequence preferably is stablyintegrated into the nuclear genome of the cell. The heterologous nucleicacid sequence preferably is retained in the cellular genome (and theheterologous nucleic acid sequence, upon expression, preferably producesa non-adenoviral gene product complementing in trans for a deficiency inat least one essential gene function of one or more regions of anadenoviral genome) for at least about 10, more preferably at least about20, passages in culture (e.g., at least about 30, 40, 100, or morepassages). Not to adhere to any particular theory, it is believed thatgenomic integration of the heterologous nucleic acid sequence encodingthe complementing factor is required to generate stable cell lines foradenoviral vector production. Additionally, complementation by transienttransfection employs both labor-intensive and inconsistent techniques,resulting in low adenovirus yield and difficulty associated withlarge-scale viral production. The introduction and stable integration ofthe heterologous nucleic acid into the genome of the cell requiresstandard molecular biology techniques that are well within the skill ofthe art, such as those described in Sambrook et al., Molecular Cloning,a Laboratory Manual, 2d ed., Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989), Watson et al., Recombinant DNA, 2d ed., ScientificAmerican Books (1992), and Ausubel et al., Current Protocols inMolecular Biology, Wiley Interscience Publishers, NY (1995).

The “heterologous nucleic acid sequence” can be any nucleic acidsequence that is not obtained from, derived from, or based upon anaturally occurring nucleic acid sequence of the precursor or host cell(i.e., the cell that is modified with the incorporation of theheterologous nucleic acid sequence to form the basis of the inventivecell). By “naturally occurring” is meant that the nucleic acid sequencecan be found in nature and has not been synthetically modified. Theheterologous nucleic acid sequence also is not obtained from, derivedfrom, or based upon an adenoviral nucleic acid sequence. For example,the heterologous nucleic acid sequence can be a viral, bacterial, plant,or animal nucleic acid sequence. A sequence is “obtained” from a sourcewhen it is isolated from that source. A sequence is “derived” from asource when it is isolated from a source but modified in any suitablemanner (e.g., by deletion, substitution (mutation), insertion, or othermodification to the sequence) so as not to disrupt the normal functionof the source gene. A sequence is “based upon” a source when thesequence is a sequence more than about 70% homologous (preferably morethan about 80% homologous, more preferably more than about 90%homologous, and most preferably more than about 95% homologous) to thesource but obtained through synthetic procedures (e.g., polynucleotidesynthesis, directed evolution, etc.). Determining the degree ofhomology, including the possibility for gaps, can be accomplished usingany suitable method (e.g., BLASTnr, provided by GenBank).Notwithstanding the foregoing, the nucleic acid sequence that makes upthe heterologous nucleic acid sequence can be naturally found in thehost cell, but located at a nonnative position within the cellulargenome and/or operably linked to a nonnative promoter.

Any suitable heterologous nucleic acid sequence that encodes anon-adenoviral gene product which complements for a deficiency in anadenoviral essential gene function can be used in the context of theinvention. The heterologous nucleic acid sequence desirably is an animalnucleic sequence (e.g., a human or murine nucleic acid sequence,especially such a nucleic acid sequence that encodes a cellular protein)or a viral nucleic acid sequence (e.g., a viral nucleic acid sequenceobtained from, derived from, or based upon CMV, EBV, HPV, or herpessimplex virus (HSV)).

The identification of heterologous nucleic acid sequences that encodenon-adenoviral gene products which complement for a deficiency in anadenoviral essential gene function is well within the skill of the art.In particular, the ordinarily skilled artisan can cotransfect cells thatdo not normally express any adenoviral gene products with an expressionconstruct comprising the heterologous nucleic acid sequence and aconstruct comprising a reporter gene (e.g., chloramphenicolacetyltransferase (CAT)) whose expression is dependent (directly orindirectly) on the presence of an essential adenoviral gene product,e.g., whose expression is regulated by an adenoviral E1A-responsivepromoter such as the E1B or E2A promoter (Spergel et al., J. Virol., 66,1021-1030 (1992)). Expression of the reporter gene, which can bedetermined by measuring reporter gene activity, indicates that thenon-adenoviral gene product produced by the heterologous nucleic acidsequence complements for a deficiency in an adenoviral essential genefunction, e.g., the transcription transactivating function of the E1Aregion. Moreover, the ordinarily skilled artisan can determine whetherthe non-adenoviral gene product transforms cells through transfectionexperiments as described by Kimura et al., supra. Other experimentsinvolving only standard molecular biology techniques, such as thosedescribed in Sambrook et al., supra, can be performed to determinewhether the non-adenoviral gene product complements for a deficiency inother adenoviral essential gene functions.

Examples of suitable heterologous nucleic acid sequences include viraland cellular nucleic acid sequences encoding a non-adenoviral geneproduct that complements for a deficiency in an adenoviral essentialgene function in the E1A region of a replication-deficient adenoviralgenome. Preferred heterologous nucleic acid sequences encoding anon-adenoviral gene product that complements for a deficiency in anessential gene function of the E1A region include the immediate early(IE) region genes I and II of human CMV, the E7 gene of HPV 16, and EBVnucleic acid sequences, as well as nucleic acids of the human HepG2 cellline, the mouse F9 teratocarcinoma stem cell line, and the mouse PCC4teratocarcinoma stem cell line (see, e.g., Spergel et al., Imperiale etal., and La Thangue et al., supra). Other examples of heterologousnucleic acid sequences include viral and cellular nucleic acid sequencesencoding a non-adenoviral gene product that complements for a deficiencyin an essential gene function in the E1B region of areplication-deficient adenoviral genome. Preferred heterologous nucleicacid sequences encoding a non-adenoviral gene product that complementsfor a deficiency in an essential gene function of the E1B region,particularly the E1B-19 kD protein and/or the E1B-55 kD protein, includenucleic acid sequences encoding the Bcl-2 protein (see, e.g., Rao etal., supra). Moreover, certain cells contain nucleic acid sequences thatendogenously complement for adenoviral E1B essential gene functiondeficiencies, and the heterologous nucleic acid sequences can be thosecellular nucleic acid sequences that provide such complementation,including HEK cells (see, e.g., Bernards et al., Virology, 150, 126-139(1986)), A549 cells (ATCC No. CCL-185), IMR90 fibroblast cells (ATCC No.CCL-186) (see, e.g., Hay et al., Human Gene Ther., 10, 579-590 (1999)),H460 cells (ATCC No. HTB-177) (see, e.g., Lee et al., Int. J. Cancer,88, 454-463 (2000)), and HCT116 cells (ATCC No. HCL-247) (see, e.g.,Ries et al., Nature Medicine, 6, 1128-1133 (2000)). Moreover, aheterologous nucleic acid sequence encoding a non-adenoviral geneproduct that complements for a deficiency in an adenoviral essentialgene function in the E1B-55 kD protein can be used to complement theoverlapping functions of the E4-ORF6 protein (see, e.g., Goodrum et al.,J. Virology, 72, 9479-9490 (1998)). Further examples of heterologousnucleic acid sequences include viral and cellular nucleic acid sequencesencoding a non-adenoviral gene product that complements for a deficiencyin an essential gene function of the E4 region of areplication-deficient adenoviral genome. Preferred heterologous nucleicacid sequences encoding a non-adenoviral gene product that complementsfor a deficiency in an essential gene function of the E4 region includenucleic acid sequences encoded by CMV. In particular, the non-adenoviralgene product can complement for a deficiency in an essential genefunction of the E4 region that is not shared by an essential genefunction of the E1B region.

The heterologous nucleic acid sequence, however, is not limited to theseexemplary sequences. Indeed, genetic sequences can vary betweendifferent animal and viral species and strains, and this natural scopeof allelic variation is included within the scope of the invention. Oncea candidate heterologous nucleic acid sequence (e.g., a CMV IE1 and/orIE2 gene region) is identified, other heterologous nucleic acidsequences encoding a non-adenoviral gene product with similar activitycan be obtained by searching the myriad of available genetic sequencedatabases that enable DNA sequence searching based on homology. One suchdatabase is the GenBank sequence database provided by the NationalCenter for Biotechnology Information (NCBI). Preferably, theheterologous sequence comprises a nucleic acid sequence which exhibitsat least about 75%, desirably at least about 85%, and more preferably atleast about 95%, nucleic acid sequence identity to (e.g., at least 97%identity to, or 100% identity with) any of the heterologous nucleicacids described herein. Determining the degree of homology, includingthe possibility for gaps, can be accomplished using any suitable method(e.g., BLASTnr, provided by GenBank).

In addition to searching sequence databases, a candidate heterologousnucleic acid sequence encoding a non-adenoviral gene product can be usedas a probe to identify homologous sequences from a genetic library viahybridization. An appropriate homologous sequence encodes anon-adenoviral gene product that functions similarly, if notidentically, to the non-adenoviral gene product encoded by the candidateheterologous nucleic acid sequence. A suitable heterologous nucleic acidsequence is that which hybridizes to a reference nucleic acid sequence(e.g., a CMV IE1 and/or IE2 gene region) under at least moderate,preferably high, stringency conditions. Exemplary moderate stringencyconditions include overnight incubation at 37° C. in a solutioncomprising 20% formamide, 5×SSC (150 mM NaCl and 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,followed by washing in 1×SSC at about 37-50° C., or substantiallysimilar conditions, e.g., the moderately stringent conditions describedin Sambrook et al., supra. High stringency conditions are conditionsthat, for example (1) use low ionic strength and high temperature forwashing, such as with a composition comprising 0.015 M sodium chlorideand 0.0015 M sodium citrate, and 0.1% sodium dodecyl sulfate (SDS) at50° C., (2) employ a denaturing agent during hybridization, such as acomposition comprising formamide, for example, 50% (v/v) formamide with0.1% bovine serum albumin (BSA), 0.1% Ficoll, 0.1% polyvinylpyrrolidone(PVP), and 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodiumchloride and 75 mM sodium citrate at 42° C., or (3) employ a compositioncomprising 50% formamide, 5×SSC (0.75 M NaCl and 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, and sonicated salmon sperm DNA (50 μg/ml), 0.1%SDS, and 10% dextran sulfate at 42° C., with washes at (i) 42° C. in0.2×SSC, (ii) at 55° C. in 50% formamide, and (iii) at 55° C. in 0.1×SSC(preferably in combination with EDTA). Additional details andexplanation of stringency of hybridization reactions are provided in,e.g., Ausubel et al., supra.

Moreover, the heterologous nucleic acid sequence can include one or moremutations (e.g., point mutations, deletions, insertions, etc.) from acorresponding naturally occurring heterologous nucleic acid sequence.Thus, where mutations are introduced in the nucleic acid sequence toeffect one or more amino acid substitutions in an encoded non-adenoviralgene product, such mutations desirably effect such amino acidsubstitutions whereby codons encoding positively-charged residues (H, K,and R) are substituted with codons encoding positively-charged residues,codons encoding negatively-charged residues (D and E) are substitutedwith codons encoding negatively-charged residues, codons encodingneutral polar residues (C, G, N, Q, S, T, and Y) are substituted withcodons encoding neutral polar residues, and codons encoding neutralnon-polar residues (A, F, I, L, M, P, V, and W) are substituted withcodons encoding neutral non-polar residues. Such mutations can also beintroduced to effect one or more amino acid substitutions in the N- orC-terminus of the encoded non-adenoviral gene product.

The heterologous nucleic acid sequence can be any suitable nucleic acidsequence as described herein that, upon expression, produces one or moregene products that complement for one or more deficiencies in anyadenoviral essential gene functions (i.e., functions necessary foradenovirus propagation). By “complements for a deficiency in anessential gene function of an adenoviral genome” is meant that the geneproduct encoded by the heterologous nucleic acid sequence exhibits anadenoviral gene function that is essential (i.e., necessary) for anadenoviral vector to propagate in a cell. For example, thenon-adenoviral gene product can induce transcription of promotersregulated by the E1A protein, such as the E2A promoter.

The non-adenoviral gene product can be an RNA sequence or a protein(e.g., a peptide or a polypeptide). Preferably, the non-adenoviral geneproduct is a protein. By “non-adenoviral gene product” is meant that thegene product exhibits less than about 50% (preferably less than about30%, more preferably less than about 10%, and most preferably less thanabout 1%) homology to a gene product encoded by an adenovirus(preferably an adenovirus of serotype 2 or 5). The degree of homologycan be determined using any suitable method known in the art (e.g.,BLAST programs).

The heterologous nucleic acid preferably encodes a full-lengthnon-adenoviral gene product, especially a non-adenoviral protein.Alternatively, the heterologous nucleic acid encodes a functionalportion of a non-adenoviral gene product, especially a protein. A“functional portion” is any portion of a non-adenoviral gene productthat complements for a deficiency in an adenoviral essential genefunction at a measurable level. A functional portion of a non-adenoviralgene product can be identified using any suitable method known in theart, such as the transfection experiments described herein.

The heterologous nucleic acid sequence, upon expression, produces atleast one non-adenoviral gene product that provides an adenoviralessential gene function, i.e., that complements in trans for one or moredeficiencies in any adenoviral essential gene function (i.e., a functionthat is necessary for adenovirus propagation). The heterologous nucleicacid sequence, upon expression, can produce a non-adenoviral geneproduct that complements for two or more deficiencies in adenoviralessential gene functions (from the same or different regions of theadenoviral genome). The heterologous nucleic acid sequence, uponexpression, can produce two or more non-adenoviral gene products, eachof which complements for a deficiency (i.e., at least one deficiency,including but not limited to, two or more deficiencies) in adenoviralessential gene functions (from the same or different regions of theadenoviral genome).

Essential adenoviral gene functions are those gene functions that arerequired for propagation (i.e., replication) of a replication-deficientadenoviral vector. Essential gene functions are encoded by, for example,the adenoviral early regions (e.g., the E1, E2, and E4 regions), lateregions (e.g., the L1-L5 regions), genes involved in viral packaging(e.g., the IVa2 gene), and viral-associated RNAs (e.g., VA-RNA I and/orVA-RNA II). Thus, the non-adenoviral gene product complements for adeficiency in at least one adenoviral essential gene function encoded bythe early regions, late regions, viral packaging regions,viral-associated RNA regions, or combinations thereof, including alladenoviral functions (e.g., to enable propagation of adenoviralamplicons comprising only inverted terminal repeats (ITRs) and thepackaging signal or only ITRs and an adenoviral promoter).

The non-adenoviral gene product desirably complements for a deficiencyin at least one essential gene function of one or more regions of theadenoviral genome selected from the early regions, e.g., the E1, E2, andE4 regions. Preferably, the non-adenoviral gene product complements intrans for a deficiency in at least one essential gene function of the E1region of the adenoviral genome. More preferably, the non-adenoviralgene product complements in trans for a deficiency in at least oneessential gene function of an adenoviral E1A coding sequence and/or anadenoviral E1B coding sequence (which together comprise the E1 region).In that respect, one non-adenoviral gene product can complement in transfor a deficiency in at least one essential gene function of the E1Acoding sequence and another (i.e., different) non-adenoviral geneproduct can complement in trans for a deficiency in at least oneessential gene function of the E1B coding sequence. In addition oralternatively to the non-adenoviral gene product(s) complementing intrans for the aforementioned deficiencies in adenoviral essential genefunctions, the same or different non-adenoviral gene product(s) cancomplement for a deficiency in at least one essential gene function ofthe E2(particularly the adenoviral DNA polymerase and terminal protein)and/or E4 regions of the adenoviral genome. Desirably, a cell thatcomplements for a deficiency in the E4 region comprises the E4-ORF6 genesequence and produces the E4-ORF6 protein. Such a cell desirablycomprises at least ORF6 and no other ORF of the E4 region of theadenoviral genome.

Although primary cells are acceptable for use as complementing celllines, the invention further provides a transformed human cellcomprising a heterologous nucleic acid sequence, which upon expressionproduces at least one non-adenoviral gene product that complements intrans for a deficiency in at least one essential gene function of one ormore regions of an adenoviral genome so as to propagate areplication-deficient adenoviral vector comprising an adenoviral genomedeficient in the at least one essential gene function of the one or moreregions when present in the cell.

The cell is “transformed” in that the cell has the ability to replicateindefinitely in culture. The human transformed cells are advantageousover primary cells for generating complementing cell lines in somerespects. In particular, transformation of primary cells with the E1transcription unit may result in an E1 expression pattern that isoptimal for transformation, but not complementation. Moreover,expression of the non-adenoviral gene product may be sufficient forcomplementation, but not transformation. In contrast, the use oftransformed cells eliminates any uncertainty related to the transformingability of a given gene product, and allows the skilled artisan todirectly determine complementation by the non-adenoviral gene product.

The transformed human cell can be any suitable such cell that comprisesa genome capable of incorporating and preferably retaining theheterologous nucleic acid encoding at least one non-adenoviral geneproduct that complements in trans for a deficiency in at least oneadenoviral essential gene function. Preferably, the cell can produceadenoviral vectors and/or adeno-associated viral (AAV) vectors wheninfected with such vectors or with nucleic acid sequences encoding theadenoviral genome. Most preferably, the cell can produce areplication-deficient adenoviral vector upon infection with the virus ortransfection with the viral genome. Particularly desirable cell typesare those that support high levels of adenovirus propagation, withdesired viral particles per cell and/or focus forming units per cellvalues as described herein with respect to the inventive cell with acellular genome comprising the heterologous nucleic acid sequence.

Preferably, the cells are, or are derived from, anchorage dependentcells, but which have the capacity to grow in suspension cultures.Examples of suitable human transformed cells include HEK-293 cells,SW-13 cells, MCF7 cells, and lung carcinoma cells such as thosedescribed herein with respect to the inventive cell with a cellulargenome comprising the heterologous nucleic acid sequence. Mostpreferably, the cell is selected from the group consisting of an A549cell, an NCI-H1299 cell, a Calu-1 cell, and an NCI-H460 cell.Alternatively, the cell need not be a lung carcinoma. In this respectthe cell is preferably a HeLa cell or an ARPE-16/HPV-16 cell. Inaddition, the human transformed cell can be any human cell transformedby a viral gene isolated from a non-adenovirus family member, such as,for example, genes encoded by Papillomaviridae, Poxviridae,Polyomaviridae, Hepadnaviridae, Picorniviridae, Flaviviridae, or anyother suitable virus family as defined by van Regenmortel et al., eds.,Virus Taxonomy, Seventh Report on the International Committee onTaxonomy of Viruses, 2000. The cell, however, is not limited to thesespecific examples. Indeed, the cell can be derived from, obtained from,or based upon any suitable human transformed cell.

The human transformed cell can comprise one heterologous nucleic acidsequence as described herein or more than one heterologous nucleic acidsequence as described herein (i.e., two or more of the heterologousnucleic acid sequence). The heterologous nucleic acid sequence can beintegrated into the cellular genome or can be otherwise present in thecell. Desirably, the heterologous nucleic acid sequence is integratedinto the cellular genome as described herein with respect to theinventive cell with a cellular genome comprising the heterologousnucleic acid sequence. When the heterologous nucleic acid sequence isnot integrated into the cellular genome, the heterologous nucleic acidsequence can reside, for example, on a plasmid, liposome, or any othertype of molecule that can harbor a heterologous nucleic acid sequenceextrachromosomally. The transformed human cell can comprise one or moreheterologous nucleic acid sequences in the cellular genome and one ormore heterologous nucleic acid sequences that are not incorporated intothe cellular genome. The descriptions of the heterologous nucleic acidsequence, non-adenoviral gene product, and complementation ofdeficiencies in adenoviral essential gene functions as described hereinwith respect to the inventive cell with a cellular genome comprising theheterologous nucleic acid sequence also apply to those same features ofthe human transformed cell.

Although not preferred, a helper virus can be provided to the inventivecell with a cellular genome comprising the heterologous nucleic acidsequence or the inventive human transformed cell in the event thateither cell does not complement for all deficiencies in essential genefunctions of the adenoviral genome of the adenoviral vector to bepropagated. The helper virus contains coding sequences that, uponexpression, produce gene products which provide in trans those genefunctions that are necessary for adenoviral propagation (e.g., the IVa2gene function). In other words, the helper virus can comprise anyadenoviral nucleic acid sequence that is not required in cis (e.g., theITRs and packaging signal) for propagation.

Both the inventive cell with a cellular genome comprising theheterologous nucleic acid sequence and the inventive human transformedcell can further comprise an “enhancing” heterologous nucleic acidsequence which upon expression produces at least one non-adenoviral geneproduct that enhances propagation of a replication-deficient adenoviralvector without necessarily complementing for a deficiency in anadenoviral essential gene function, so as to propagate morereplication-deficient adenoviral vectors when present in the cell thanwhen the “enhancing” heterologous nucleic acid sequence is absent fromthe cell. Although genomic integration of this “enhancing” heterologousnucleic acid sequence is preferred, the “enhancing” heterologous nucleicacid sequence also can be maintained in the cell extrachromosomally(e.g., on a plasmid).

The “enhancing” heterologous nucleic acid sequence can encode, forexample, a protein that inhibits and/or prevents apoptosis (e.g.,Bcl-2). Moreover, the “enhancing” heterologous nucleic acid sequence canencode, for example, an RNA molecule or protein that improves theefficiency or rate of replication-deficient adenoviral vectorpropagation.

The expression of any of the heterologous nucleic acid sequences in theinventive cell with a cellular genome comprising the heterologousnucleic acid sequence or the inventive transformed human cell iscontrolled by a suitable expression control sequence operably linked tothe heterologous nucleic acid sequence. An “expression control sequence”is any nucleic acid sequence that promotes, enhances, or controlsexpression (typically and preferably transcription) of another nucleicacid sequence. Suitable expression control sequences includeconstitutive promoters, inducible promoters, repressible promoters, andenhancers. The heterologous nucleic acid sequence can be regulated byits endogenous promoter or by a non-native promoter sequence. Examplesof suitable non-native promoters include the CMV immediate earlypromoter, the phosphoglycerate kinase (PGK) promoter, the long terminalrepeat promoter of the Rous sarcoma virus (LTR-RSV), the sheepmetallothionien promoter, and the human ubiquitin C promoter.Alternatively, expression of the heterologous nucleic acid sequence canbe controlled by a chimeric promoter sequence. The promoter sequence is“chimeric” when it comprises at least two nucleic acid sequence portionsobtained from, derived from, or based upon at least two differentsources (e.g., two different regions of an organism's genome, twodifferent organisms, or an organism combined with a synthetic sequence).In addition, the expression control sequence can be activated uponinfection with a viral vector, such as a replication-deficientadenoviral vector, or contact with viral peptides. When the nucleic acidsequence that makes up the heterologous nucleic acid sequence isnaturally found in the host cell but operably linked to a nonnativepromoter, the nonnative promoter can be introduced into the inventivecell by homologous recombination (see, e.g., U.S. Pat. No. 5,641,670) orby random promoter insertion (see, e.g., Harrington et al., NatureBiotechnology, 19, 440-445 (2001)). Suitable expression controlsequences can be determined using eukaryotic expression systems such asare generally described in Sambrook et al., supra, and by using reportergene systems (see, e.g., Taira et al., Gene, 263, 285-292 (2001)).

The invention also provides a system comprising the inventive cell and areplication-deficient adenoviral vector comprising an adenoviral genomedeficient in the at least one essential gene function of the one or moreregions (i.e., a replication-deficient adenoviral vector comprising thedeficiencies complemented for by the inventive cell). The inventionfurther provides a method of propagating a replication-deficientadenoviral vector. The method comprises providing a cell of theinvention, introducing the replication-deficient adenoviral vector intothe cell, wherein the replication-deficient adenoviral vector comprisesan adenoviral genome deficient in the at least one essential genefunction of the one or more regions, and maintaining the cell (e.g.,under conditions suitable for adenoviral propagation) to propagate theadenoviral vector.

The adenoviral vector is deficient in at least one gene function (of theadenoviral genome) required for viral propagation (i.e., an adenoviralessential gene function), thereby resulting in a “replication-deficient”adenoviral vector. The adenoviral vector is deficient in the one or moreadenoviral essential gene functions complemented for by the inventivecell to allow for propagation of the replication-deficient adenoviralvector when present in the cell.

Preferably, the adenoviral vector is deficient in at least one essentialgene function of the E1 region, e.g., the E1a region and/or the E1bregion, of the adenoviral genome that is required for viral replication.The recombinant adenovirus also can have a mutation in the major latepromoter (MLP), as discussed in International Patent Application WO00/00628. More preferably, the vector is deficient in at least oneessential gene function of the E1 region and at least part of thenonessential E3 region (e.g., an Xba I deletion of the E3 region). Theadenoviral vector can be “multiply deficient,” meaning that theadenoviral vector is deficient in one or more essential gene functionsin each of two or more regions of the adenoviral geonome. For example,the aforementioned E1-deficient or E1-, E3-deficient adenoviral vectorscan be further deficient in at least one essential gene of the E4 regionand/or at least one essential gene of the E2 region (e.g., the E2Aregion and/or E2B region). Adenoviral vectors deleted of the entire E4region can elicit lower host immune responses. Examples of suitableadenoviral vectors include adenoviral vectors that lack (a) all or partof the E1 region and all or part of the E2 region, (b) all or part ofthe E1 region, all or part of the E2 region, and all or part of the E3region, (c) all or part of the E1 region, all or part of the E2 region,all or part of the E3 region, and all or part of the E4 region, (d) atleast part of the E1a region, at least part of the E1b region, at leastpart of the E2a region, and at least part of the E3 region, (e) at leastpart of the E1 region, at least part of the E3 region, and at least partof the E4 region, and (f) all essential adenoviral gene products (e.g.,adenoviral amplicons comprising ITRs and the packaging signal only). Theadenoviral vector can contain a wild type pIX gene. Alternatively,although not preferably, the adenoviral vector also can contain a pIXgene that has been modified by mutation, deletion, or any suitable DNAmodification procedure.

The replication-deficient adenoviral vector can be generated by usingany species, strain, subtype, or mixture of species, strains, orsubtypes, of an adenovirus or a chimeric adenovirus as the source ofvector DNA. The adenoviral vector can be any adenoviral vector capableof growth in a cell, which is in some significant part (although notnecessarily substantially) derived from or based upon the genome of anadenovirus. The adenoviral vector preferably comprises an adenoviralgenome of a wild-type adenovirus of group C, especially of serotype(i.e., Ad5). Adenoviral vectors are well known in the art and aredescribed in, for example, U.S. Pat. Nos. 5,559,099, 5,712,136,5,731,190, 5,837,511, 5,846,782, 5,851,806, 5,962,311, 5,965,541,5,981,225, 5,994,106, 6,020,191, and 6,113,913, International PatentApplications WO 95/34671, WO 97/21826, and WO 00/00628, and ThomasShenk, “Adenoviridae and their Replication,” and M. S. Horwitz,“Adenoviruses,” Chapters 67 and 68, respectively, in Virology, B. N.Fields et al., eds., 3d ed., Raven Press, Ltd., New York (1996).

The construction of adenoviral vectors is well understood in the art andinvolves the use of standard molecular biological techniques, such asthose described in, for example, Sambrook et al., supra, Watson et al.,supra, Ausubel et al., supra, and other references mentioned herein.Moreover, adenoviral vectors can be constructed and/or purified usingthe methods set forth, for example, in U.S. Pat. No. 5,965,358 andInternational Patent Applications WO 98/56937, WO 99/15686, and WO99/54441.

When the cell is used to propagate a replication-deficient adenoviralvector, it is desirable to avoid a recombination event between thecellular genome (of the cell) and the adenoviral genome (of theadenoviral vector) that would result in the generation of areplication-competent adenovirus (RCA). As such, there is preferablyinsufficient overlap between the genome of the cell and thereplication-deficient adenoviral vector genome to mediate arecombination event sufficient to result in a replication-competentadenovirus. If overlap exists, the overlapping sequences desirably arepredominantly located in the nucleic acid flanking the coding region ofthe complementation factor (the “trans-complementing region”) in thecellular genome and the nucleotide sequences adjacent to the missingregion(s) of the adenoviral genome. Ideally, there is no overlap betweenthe cellular genome and the adenoviral vector genome. However, it isacceptable that partial overlap exists between the cellular genome andthe adenoviral vector genome on one side of the trans-complementingregion. In such an event, the region of homology preferably iscontiguous with the trans-complementing region. For example, when thecell comprises a trans-complementing region comprising a nucleotidesequence of the adenoviral E1 region, the cell desirably lackshomologous sequences on the 5′side (left side) of thetrans-complementing region corresponding to the adenoviral invertedterminal repeats (ITRs) and packaging signal sequences, but containshomologous sequences on the 3′side (right side) of thetrans-complementing region. The region of homology is at least about2000 base pairs, preferably at least about 1000 base pairs (e.g., atleast about 1500 base pairs), more preferably at least about 700 basepairs, and most preferably at least about 300 base pairs. The generationof RCA desirably is diminished such that (a) the cell produces less thanabout one replication-competent adenoviral vector for at least about 20passages after infection with the adenoviral vector, (b) the cellproduces less than about one replication-competent adenoviral vector ina period of about 36 hours post-infection, (c) the cell produces lessthan about one replication-competent adenoviral vector per 1×10¹⁰ totalviral particles (preferably 1×10¹¹ total viral particles, morepreferably 1×10¹² total viral particles, and most preferably 1×10¹³total viral particles), or any combination of (a)-(c). Optimally, theamount of overlap between the cellular genome and the adenoviral genome(i.e., the genome of the adenoviral vector being propagated in the cell)is insufficient to mediate a homologous recombination event that resultsin a replication-competent adenoviral vector such thatreplication-competent adenoviruses are eliminated from the vector stocksresulting from propagation of the replication-deficient adenoviralvector in the cell. Virus growth yield and virus plaque formation havebeen previously described (see, e.g., Burlseson et al., Virology: aLaboratory Manual, Academic Press Inc. (1992)), and measuring RCA as afunction of plaque forming units is described in U.S. Pat. 5,994,106.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example describes the construction of a primary cell having acellular genome comprising the CMV immediate early gene regions 1 and 2heterologous nucleic acid sequences, which, upon expression, produce atleast one non-adenoviral protein (i.e., the IE1 and IE2 proteins).

Plasmid pCMVXbaEpuro contains the XbaI-E fragment of HCMV DNA (0.68-0.77map units), which includes the CMV immediate early (IE) regions 1 and 2inserted into the NruI-ApaI digested pSMTpuro-ORF6 plasmid. ThepSMTpuro-ORF6 plasmid contains the adenovirus 5 E4-ORF6 gene under thecontrol of the sheep metallothionein promoter (see, e.g., InternationalPatent Application WO 95/34671).

Primary human embryonic lung (HEL) cells are cultured in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% calf serum, 100 μgof penicillin per milliliter (all components from Life Technologies,Gaithersburg, Md.). The HEL cells are then transfected with pCMVXbaEpuroby the calcium phosphate method (Sambrook et al., supra). Followingtransfection, 2.5 μg/ml of puromycin is added to the culture medium forselection of the CMV IE1 and IE2 expressing cells, which are clonallyisolated and propagated (see Sambrook et al., supra). Approximately 24hours post-transfection, expression of CMV IE1 and IE2 genes is assayedvia Northern and Western blotting. Integration of the CMV sequences isconfirmed by Southern blotting.

EXAMPLE 2

This example describes a method for demonstrating the ability of aninventive cell having a cellular genome comprising a heterologousnucleic acid sequence encoding the CMV IE1 and IE2 proteins to supportpropagation and production of a replication-deficient adenoviral vector.

The HEL cells of Example 1 are cultured using routine tissue culturetechniques. Monolayers at passages 5 and 10 are screened for E1Acomplementation by a virus production assay (see, e.g., Burlseson etal., Virology: A Laboratory Manual, Academic Press Inc. (1992)). In thatrespect, the cells are infected with a replication-deficient adenoviralvector wherein the E1A region has been deleted from the adenoviralgenome thereof. Specifically, the cells are infected with anE1A-deficient adenoviral vector, which contains the E1B region encodingthe protein AdE 1B, at a multiplicity of infection (MOI) of 10. Celllysates are prepared at 3 days post-infection (d.p.i.), and the amountof active virus in the lysates is determined by a focal forming unit(FFU) assay (Cleghorn et al., Virology, 197, 564-575 (1993)). Thedetection of significant yields of AdE1B for the cells at passages 5 and10 evidences the ability of the cells to complement in trans fordeficiencies in adenoviral essential gene functions of the E1A region ofthe adenoviral genome.

EXAMPLE 3

This example describes the construction of a transformed human cellhaving a cellular genome comprising the CMV immediate early gene regions1 and 2 heterologous nucleic acid sequences, which, upon expression,produce at least one non-adenoviral protein (i.e., the IE1 and IE2proteins).

HeLa cells (ATCC CCL-2) are cultured in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% calf serum, 100 μg of penicillin permilliliter (all components from Life Technologies, Gaithersburg, Md.).The HeLa cells are then transfected with the pCMVXbaEpuro plasmid ofExample 1 by the calcium phosphate method (Sambrook et al., supra).Following transfection, 2.5 μg/ml of puromycin is added to the culturemedium for selection of the CMV IE1 and IE2 expressing cells, which areclonally isolated and propagated (see Sambrook et al., supra).Approximately 24 hours post-transfection, expression of CMV IE1 and IE2genes is assayed via Northern and Western blotting. Integration of theCMV sequences is confirmed by Southern blotting.

EXAMPLE 4

This example describes a method for demonstrating the ability of aninventive cell having a cellular genome comprising a heterologousnucleic acid sequence encoding the

CMV IE1 and IE2 proteins to support propagation and production of areplication-deficient adenoviral vector.

The HeLa cells of Example 3 are cultured using routine tissue culturetechniques. Monolayers at passages 5 and 10 are screened for E1Acomplementation by a virus production assay (see, e.g., Burlseson etal., Virology: A Laboratory Manual, Academic Press Inc. (1992)). In thatrespect, the cells are infected with a replication-deficient adenoviralvector wherein the E1A region has been deleted from the adenoviralgenome thereof. Specifically, the cells are infected with anE1A-deficient adenoviral vector, which contains the E1B region encodingthe protein AdE1B, at a multiplicity of infection (MOI) of 10. Celllysates are prepared at 3 days post-infection (d.p.i.), and the amountof active virus in the lysates is determined by a focal forming unit(FFU) assay (Cleghom et al., Virology, 197, 564-575 (1993)). Thedetection of significant yields of AdE1B for the cells at passages 5 and10 evidences the ability of the cells to complement in trans fordeficiencies in adenoviral essential gene functions of the E1A region ofthe adenoviral genome.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations of those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventors expect skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than as specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

What is claimed is:
 1. A human retinal cell comprising at least one heterologous nucleic acid sequence which upon expression produces at least one non-adenoviral gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome selected from the group consisting of the E1, E2A, and E4 regions so as to propagate a replication-deficient adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the E1, E2A, and/or E4 regions when present in the human retinal cell.
 2. The human retinal cell of claim 1, wherein at least one non-adeno viral gene product complements in trans for a deficiency in the E1A region of an adenoviral genome.
 3. The human retinal cell of claim 1, wherein at least one non-adenoviral gene product complements in trans for a deficiency in E4-ORF6 of an adenoviral genome.
 4. The human retinal cell of claim 1, wherein at least one non-adenoviral gene product is a viral protein.
 5. The human retinal cell of claim 1, wherein at least one non-adenoviral gene product is a cellular protein.
 6. The human retinal cell of claim 1, wherein the human retinal cell further comprises a heterologous nucleic acid sequence which upon expression produces a non-adenoviral gene product that enhances propagation of the replication-deficient adenoviral vector, so as to produce more replication-deficient adenoviral vectors when the heterologous nucleic acid sequence is present in the human retinal cell than when it is absent from the human retinal cell.
 7. A method of propagating a replication-deficient adenoviral vector, which method comprises: (a) providing the human retinal cell of claim 1, (b) introducing a replication-deficient adenoviral vector into the human retinal cell, wherein the replication-deficient adenoviral vector comprises an adenoviral genome deficient in the at least one essential gene function of the E1, E2A, and/or E4 regions, and (c) maintaining the human retinal cell to propagate the replication-deficient adenoviral vector.
 8. The human retinal cell of claim 1, wherein the human retinal cell is a human embryonic retinal cell.
 9. The human retinal cell of claim 1, wherein the at least one heterologous nucleic acid sequence is integrated into the cellular genome.
 10. The method of claim 7, wherein at least one non-adenoviral gene product complements in trans for a deficiency in the E1A region of an adenoviral genome.
 11. The method of claim 7, wherein at least one non-adenoviral gene product complements in trans for a deficiency in E4-ORF6 of an adenoviral genome.
 12. The method of claim 7, wherein at least one non-adenoviral gene product is a viral protein.
 13. The method of claim 7, wherein at least one non-adenoviral gene product is a cellular protein.
 14. The method of claim 7, wherein the human retinal cell is a human embryonic retinal cell.
 15. The method of claim 7, wherein the at least one heterologous nucleic acid sequence is integrated into the cellular genome.
 16. A lung carcinoma cell comprising at least one heterologous nucleic acid sequence which upon expression produces at least one non-adenoviral gene product that complements in trans for a deficiency in at least one essential gene function of the E1 region of an adenoviral genome and a deficiency in one or more essential gene functions in either or both of the E2A region and the E4 region the adenoviral genome so as to propagate a replication-deficient adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the E1 region and either or both of the E2A region and the E4 region when present in the lung carcinoma cell.
 17. The lung carcinoma cell of claim 16, wherein at least one non-adenoviral gene product complements in trans for a deficiency in the E1A region of an adenoviral genome.
 18. The lung carcinoma cell of claim 16, wherein at least one non-adenoviral gene product complements in trans for a deficiency in E4-ORF6 of an adenoviral genome.
 19. The lung carcinoma cell of claim 16, wherein at least one non-adenoviral gene product is a viral protein.
 20. The lung carcinoma cell of claim 16, wherein at least one non-adenoviral gene product is a cellular protein.
 21. The lung carcinoma cell of claim 16, wherein the lung carcinoma cell is selected from the group consisting of an A549 cell, an NCI-H1299 cell, a Calu-1 cell, and an NC1-H460 cell.
 22. The lung carcinoma cell of claim 16, wherein the lung carcinoma cell further comprises a heterologous nucleic acid sequence which upon expression produces a non-adenoviral gene product that enhances propagation of the replication-deficient adenoviral vector, so as to produce more replication-deficient adenoviral vectors when the heterologous nucleic acid sequence is present in the lung carcinoma cell than when it is absent from the lung carcinoma cell.
 23. A method of propagating a replication-deficient adenoviral vector, which method comprises: (a) providing the lung carcinoma cell of claim 16, (b) introducing a replication-deficient adenoviral vector into the lung carcinoma cell, wherein the replication-deficient adenoviral vector comprises an adenoviral genome deficient in the at least one essential gene function of the E1 region and either or both of the E2A region and the E4 region, and (c) maintaining the lung carcinoma cell to propagate the replication-deficient adenoviral vector.
 24. The lung carcinoma cell of claim 16, wherein the at least one heterologous nucleic acid sequence is integrated into the cellular genome.
 25. The method of claim 23, wherein at least one non-adenoviral gene product complements in trans for a deficiency in the E1A region of an adenoviral genome.
 26. The method of claim 23, wherein at least one non-adenoviral gene product complements in trans for a deficiency in E4-ORF6 of an adenoviral genome.
 27. The method of claim 23, wherein at least one non-adenoviral gene product is a viral protein.
 28. The method of claim 23, wherein at least one non-adenoviral gene product is a cellular protein.
 29. The method of claim 23, wherein the lung carcinoma cell is selected from the group consisting of an A549 cell, an NCI-H1299 cell, a Calu-1 cell, and an NCI-H460 cell.
 30. The method of claim 23, wherein the at least one heterologous nucleic acid sequence is integrated into the cellular genome.
 31. A cell having a cellular genome comprising at least one heterologous nucleic acid sequence which upon expression produces at least one non-adenoviral gene product that complements in trans for a deficiency in at least one essential gene function of the E4 region of an adenoviral genome so as to propagate a replication-deficient adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the E4 region when present in the cell.
 32. The cell of claim 31, wherein the cell further comprises a heterologous nucleic acid sequence which upon expression produces at least one non-adenoviral gene product that complements in trans for a deficiency in the E1A region of an adenoviral genome.
 33. The cell of claim 31, wherein at least one non-adenoviral gene product complements in trans for a deficiency in E4-ORF6 of an adenoviral genome.
 34. The cell of claim 31, wherein at least one non-adenoviral gene product is a viral protein.
 35. The cell of claim 31, wherein at least one non-adenoviral gene product is a cellular protein.
 36. The cell of claim 31, wherein the at least one heterologous nucleic acid sequence is integrated into the cellular genome.
 37. A method of propagating a replication-deficient adenoviral vector, which method comprises: (a) providing the cell of claim 31, (b) introducing a replication-deficient adenoviral vector into the cell, wherein the replication-deficient adenoviral vector comprises an adenoviral genome deficient in the at least one essential gene function of the E4 region, and (c) maintaining the cell to propagate the replication-deficient adenoviral vector. 